Action of morpholinos in Ciona embryos

First, using morpholino against lacZ, we demonstrate that the morpholino specifically suppresses the translation of the gene introduced exogenously into Ciona eggs. Second, using morpholino against an alkaline phosphatase gene, we show that the morpholino suppresses the translation of the endogenous gene as well. Third, using morpholino against beta-catenin gene, we confirm that the suppression by the morpholino can be rescued by injection of beta-catenin mRNA. All of these results indicate that morpholino act in Ciona embryos to specifically block the function of endogenous genes as well as exogenously introduced genes. genesis 30: 103--106, 2001.     

Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.     

Construction of BAC libraries derived from the ascidian Ciona intestinalis

Large insert genomic bacterial artificial chromosome (BAC) libraries were constructed from a basal chordate, the ascidian Ciona intestinalis. Insert analyses of randomly selected clones indicated that in the first library the mean insert size was 135 kb and predicted a 15-fold coverage of the Ciona genome, and in the second library the mean insert size was 165 kb and predicted a 5-fold coverage of the genome. These first large insert genomic libraries of the ascidian should increase the speed of genomic analyses of basal chordates.     

EST analysis of gene expression in testis of the ascidian Ciona intestinalis

To explore the gene expression underlying spermatogenesis, a large-scale analysis has been done on the cDNAs from testis of the ascidian, Ciona intestinalis. A set of 5,461 expressed sequence tags was analyzed and grouped into 2,806 independent clusters. Approximately 30% of the clusters showed significant sequence matches to the proteins reported in DDBJ/GenBank/EMBL database including a set of proteins closely related to the gene regulation during spermatogenesis, functional and morphological changes of spermatogenic cells during spermiogenesis, and physiological functions of sperm, as well as those with housekeeping functions commonly expressed in other cells. Some clones show similarities to the proteins present in vertebrate lymphocytes, suggesting a primitive immune system in ascidians. We have also found some genes that are known to participate in hormonal regulation of spermatogenesis in vertebrates. The large majority of the genes expressed in Ciona testis show no significant matches to known proteins and the further analysis of these genes may shed new light on the molecular mechanism of spermatogenesis and sperm functions.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VIII. Genes for PI3K signaling and cell cycle

Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.